Manual and automated roller bottle systems have been used for over 40 years in the pharmaceutical, biochemical, and medical fields for processes such as cell growth and infection, heterologous glycoprotein production, vaccine preparation, and high density plant cell cultivation. [H. Tanaka, F. Nishijima, M. Suwa, and T. Iwamoto, Biotechnol. Bioeng. 25, 2359 (1983); H. Tanaka, Process Biochem. Aug., 106 (1987); C. Y. Hong, T. P. Labuza, and S. K. Harlander, Biotechnol. Prog. 5:4, 137 (1989); Y. A. Elliot, Bioprocess Tech. 10, 207 (1990); V. G. Kalthod, Novel Carrier and Reactor for Culture of Attachment Dependent Mammalian Cells. D. S C. Thesis. Washington University, St. Louis, Mo. (1991); E. I. Tsao, M. A. Bohn, D. R. Omstead, and M. J. Munster. Annals N.Y. Acad. Sci. 665, 127 (1992); R. Pennell and C. Milstein, J. of Immun. Meth. 146, 43 (1992); E. Olivas, B. B. D.-M. Chen, and W. S. Walker, J. Immun. Meth. 182, 73 (1995); R. Singhvi, J. F. Markusen, B. Ky, B. J. Horvath, and J. G. Aunins, Cytotechnology 22, 79 (1996); R. Kunitake, A. Suzuki, H. Ichihashi, S. Matsuda, O. Hirai, and K. Morimoto, J. Biotechnology 52:3, 289 (1997).]. Despite efforts by numerous investigators to develop unit operation based systems, such as microcarrier cultures, for the production of anchorage dependent cells or cell products [E. Van Hemert, D. G. Kilburn, and A. L. Van Wezel, Biotechnol. Bioeng. 11, 875 (1969); C. Horng and W. McLimans, Biotechnol. Bioeng. 17, 713 (1975); R. E. Spier and J. P. Whiteside, Biotechnol. Bioeng. 18, 649 (1976); D. W. Levine, D.Wang, and W. G. Thilly, Biotechnol. Bioeng. 2, 821 (1979); J. J. Clark and M. D. Hirtenstein, J. Interferon Research 1, 391 (1981); B. J. Montagnon, B. Fanget, and A. J. Nicolas, Developments in Biological Standards 47, 55 (1981); V. G. Edy, Adv. Exp. Med. Biol. 172, 169 (1984); E. Rivera, C. G. Sjosten, R. Bergman, K. A. Karlsson, Research in Veterinary Science 41, 391 (1986); and R. M. Gallegos Gallegos, E. L. Espinosa Larios, L. R. Ramirez, R. K. Schmid, and A. G. Setien, Archives in Medical Research 26:1, 59 (1995).], roller bottle systems still prevail in research and industry. Additionally, for industrial scale production of cell culture products (i.e. vaccines), cells are frequently passaged in roller bottles prior to transfer to microcarrier cultures for the final growth phase even when unit operation based systems are utilized [V. G. Edy, Adv. Exp. Med. Biol. 172, 169 (1984)].
Widespread use of the roller bottle is due to several reasons. Most notably, the process relies on very simple technology: a horizontal cylindrical vessel is filled approximately one-third full and axially rotated. Thus, scale-up development is not required, resulting in reduced developmental timelines for industry and faster introduction to market for new products. The system allows constant fluid-gas contact, and easy addition of nutrients without interruption of the process. In addition, the process is capable of maintaining sterile conditions for prolonged times, contamination of one or more roller bottles does not result in the contamination of an entire lot, precise control of nutrient and waste-product levels is possible, and the direct monitoring of the cells is relatively simple [E. I. Tsao, M. A. Bohn, D. R. Omstead, and M. J. Munster. Annals N.Y. Acad. Sci. 665, 127 (1992)].
On the other hand, roller bottles are limited in surface area available for growth and in the volume of harvest fluid obtained. Manpower and facility space requirements are higher than for unit operation systems such as microcarriers, since hundreds of roller bottles are typically operated for a single production run; although, new automation systems are addressing this issue [R. Kunitake, A. Suzuki, H. Ichihashi, S. Matsuda, O. Hirai, and K. Morimoto, J. Biotechnology 52:3, 289 (1997); and R. Archer and L. Wood, Proceedings of the 11th Annual Meeting of European Society for Animal Cell Technology, Brighton, U.K., Sep. 2-6, 1991.]. In addition, the performance of cell growth and infection is believed to be significantly reduced due to flow and mixing dynamics, perhaps by preventing infected cells from attaching to host cells attached to the bottle walls [Y. A. Elliot, Bioprocess Tech. 10, 207 (1990)]. Although these issues point toward an obvious need for flow analysis and process design criteria, there have been no published results to date on either of these topics.
The conventional method of mixing in roller bottles is efficient. The bottles are generally rotated at a uniform rate in one direction for cell planting, cell growth and/or virus propagation. A rotation frequency of 0.125 rpm to 5.0 rpm is typical. This uniform rotation, however, results in the formation of dead zones within the roller bottle where cells or other particles such as viruses are trapped in cyclic orbits, never making it to the surface of the roller bottle. At cell planting, for anchorage-dependent cells, it is important that the cells come in contact rapidly with the sides of the roller bottle, since only then can the cells become attached to the container wall and form the cell sheets. Slow attachment leads to low viability of the cells and/or inhomogeneous planting, and hence inhomogeneous growth on the roller bottle surface. During cell growth, such inefficient mixing limits cell growth because the poorly mixed medium does not supply the cells with adequate nutrients (e.g. oxygen) or remove toxins (e.g. carbon dioxide) from a submerged, surface-attached cell sheet as the bottle rotates. During propagation of many viruses, the rapidity of virus attachment to the cells is important to maintain inoculum infectivity and to achieve a rapid and homogeneous infection. Again, poor liquid mixing thwarts these goals.
The shortcomings of conventional roller bottle mixing are underscored when it is applied to virus propagation, especially for viruses where the virus inoculum to the process is an infected cell suspension. This is the case for several herpesviruses such as Marek's Disease Virus of poultry (D. Ben Nathan and S. Lustig "Production of Marek's Disease Vaccine" in Viral Vaccines, Wiley-Liss, 1990, pp. 347-365), and Varicella Zoster Virus (P. J. Provost et al. U.S. Pat. Nos. 5,360,736 and 5,607,852; and Krah et al. "Enhancement of Varicella-zoster Virus Plaquing Efficiency with an Agrarose Overlay Medium" J. Vir. Methods 27, pp.319-326 (1990).). The efficiency of virus propagation is dependent on the infectious cells coming in contact with the cell sheet, where infectious foci are created. The infection then spreads across the cell sheet from these foci. In conventional roller bottle mixing, however, many infectious cells become trapped in closed, symmetrical orbits and never reach the cell sheet on the bottle surface. In addition, conventional roller bottles display poor axial mixing, resulting in large heterogeneous areas.
The instant application describes detail mathematical and experimental characterizations of the fluid flow profiles within rotating roller bottles, including particle trajectories, fluid mixing patterns, and unsteady-state flow strategies. The results point to ways in which cell proliferation and infection can be optimized by simple modifications to the roller bottle's rotation. Thus, the instant invention describes an improved method for mixing of a varicella-infected cell culture in roller bottles that introduces cross-sectional flow perturbations. These perturbations disrupt the closed orbits that cells experience during conventional mixing and facilitate particle settling. Furthermore, this invention relates to a mixing process that ensures that cells come in contact with adequate amounts of nutrient-rich medium and by increasing the contact between the cells and the roller bottle wall or cell sheet and thereby enhances productivity.